The gastrointestinal immune system mediates contact between the host and the microbiota that symbiotically colonize this mucosal surface, while protecting the host against pathological invasion. Exposure to nutritional, microbial, and host antigens in the intestine creates an extended self that varies dynamically in this complex environment. Thus, the division between self and non-self is often indistinct, and multiple regulatory mechanisms have evolved to restrain misdirected immune responses. Failure of these regulatory pathways and/or imbalances in the composition of the microbiome can result in pathologic inflammatory processes that include inflammatory bowel disease (IBD). Among those regulatory pathways mediated by lymphocytes, Foxp3+ regulatory T (Treg) cells play a requisite role in establishing and maintaining immunological tolerance. Two subsets of Treg cells have been described. Natural Treg (nTreg) cells arise as a discrete and largely stable lineage originating in the thymus. Induced Treg (iTreg) cells are generated in situ from naive conventional T cells in the periphery. The iTreg subset of Treg cells appears to be particularly prevalent at mucosal surfaces where they may serve to control host responses to the commensal microbiota. Specific commensal microbes are associated with an increase in the frequency of pro-inflammatory Th17 effector cells within the lamina propria of the small intestine. Since both iTreg and Th17 cells can develop from naive T cells through activation in the presence of TGF-, these observations suggest an influence of the microbiome on both iTreg and Th17 cell differentiation. The developmental stability of iTreg cells in the gut and the influence of the microbiome on iTreg cell development are critical and unanswered questions in the field. The broad objective of the proposed research is to determine mechanisms that maintain immune tolerance in the gut, and characterize how mucosal inflammation and the intestinal microbiota may contribute to a break in self tolerance in this tissue. The proposed experiments will test the hypotheses that the iTreg pool arises via a flexible developmental pathway, that iTreg cells retain potential colitic specificities, and that the development and stability of the iTreg population reflects the composition of the microbiome. The proposed experiments will utilize genetic, immunological, and next-generation genomic approaches to establish the propensity of iTreg cells to adopt an alternate pathogenic fate, and determine the influence of the microbiome on IBD and the development of iTreg cells. The data will emphasize the iTreg/Th17 cell balance as an essential regulatory checkpoint. The design of therapeutic strategies for IBD may depend upon this type of information.